Proposal Abstract Prostate cancer (PCa) is the most commonly diagnosed solid tumor and the second leading cause of cancer- related deaths in U.S. males. Recently the Cramer lab discovered that a specific subtype of PCa, MAP3K7-null PCa, occurs in 30-40% of patients. MAP3K7 is a known tumor suppressor and loss of this protein is associated with an aggressive form of the disease with a poor rate of disease-free survival. Interestingly, co-suppression of tumor suppressor CHD1, a protein vital for transcriptional regulation and genomic stability, often occurs with loss of MAP3K7 leading to an even more aggressive cancer phenotype. Therefore, identifying novel therapeutic strategies for this lethal subtype of PCa can have a high impact on PCa patient survival. Cancer cells often develop apoptosis-resistance leading to the hypothesis that cancer therapy can be improved by activating an alternative mechanism of cell death such as necroptosis. Necroptosis is a kinase-driven, caspase-independent form of cell death. Necroptosis is immunogenic while apoptosis suppresses anti-tumor immune responses. Importantly, autophagy has a vital role in promoting cell death. When MAP3K7-null cells are treated with TRAIL, they undergo necroptotic cell death. Autophagosome protein p62 is crucial for switching cell death from apoptosis to necroptosis by recruitment of RIPK1 to the necrosome. However, the underlying mechanisms of autophagy-mediated cell death are poorly understood. It is unknown if autophagy only controls whether or not cells die or also promotes other aspects of programmed cell death. Our working hypothesis is that manipulation of autophagy machinery can enhance TRAIL-induced necroptosis and be an effective therapeutic option for MAP3K7-null PCa. First, we will determine the role of autophagy in TRAIL-induced necroptosis. This aim will determine interacting proteins between the autophagosome and necrosome by BioID, proximity ligation, assay, immuno electron and super-resolution microscopy, and co-immunoprecipitation. Further, we will examine degradation of pro- apoptotic and pro-survival proteins by the autophagosome upon treatment with TRAIL. Lastly, we will examine the functional roles of interacting proteins by targeted knockdown and analysis of cell viability and cell death. To determine the immunogenic effects of necroptosis, we will analyze in vivo anti-tumor effects of TRAIL. Immune compromised and immune competent mice will be treated with TRAIL for 12 weeks after tumor implantation through our novel prostate tissue recombination method. Tumor size and necroptotic, apoptotic and immune markers will be analyzed in addition to immune activation. We will then repeat this experiment with targeting of interacting proteins found in Aim 1 to enhance the anti-tumor adaptive immune response. Significantly, this research will reveal a novel therapeutic strategy for treatment of a lethal form of PCa, MAP3K7-null, which has no effective therapeutic strategies at this time. Further, it will elucidate the role of autophagy machinery in cell signaling and stimulation of necroptotic cell death.!